Bioorg. Med. Chem. Lett. 2010 Ranjit

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<ul><li><p>Bioorganic &amp; Medicinal Chemistry Letters 20 (2010) 45314534Contents lists available at ScienceDirect</p><p>Bioorganic &amp; Medicinal Chemistry Letters</p><p>journal homepage: www.elsevier .com/ locate/bmclSmall molecule functional analogs of peptides that inhibit k site-specificrecombination and bind Holliday junctions</p><p>Dev K. Ranjit a,, Marc C. Rideout a, Adel Nefzi b, John M. Ostresh c, Clemencia Pinilla c, Anca M. Segall a,*aDepartment of Biology and Center for Microbial Sciences, San Diego State University, San Diego, CA 92182-4614, USAb Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987-2352, USAc Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, 2-129, San Diego, CA 92121-1120, USA</p><p>a r t i c l e i n f o a b s t r a c tArticle history:Received 5 April 2010Revised 3 June 2010Accepted 4 June 2010Available online 8 June 2010</p><p>Keywords:Site-specific recombinationk-IntegraseHolliday junctionCombinatorial librariesRecG helicase0960-894X/$ - see front matter 2010 Elsevier Ltd. Adoi:10.1016/j.bmcl.2010.06.029</p><p>* Corresponding author. Tel.: +1 619 594 6528; faxE-mail address: (A.M. Se</p><p> Present address: Department of Pathology andNebraska Medical Center, Omaha, NE 68198-5900, USAOur lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ),central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors,WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increasethe therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activ-ities. We screened 11 small molecule libraries comprising over nine million individual compounds andidentified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specificrecombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJresolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperperme-able outer membrane; nonetheless, this is an important step in developing a functional analog of the pep-tide inhibitors.</p><p> 2010 Elsevier Ltd. All rights reserved.Site-specific recombination (SSR) catalyzed by bacteriophage kIntegrase (Int) requires the formation and subsequent resolution ofa4-armedDNAintermediateknownasaHolliday Junction (HJ) (Sup-plementary data Fig. 1A, and reviewed in Ref. 1).Wehave previouslyidentified hexapeptide inhibitors from combinatorial peptide li-braries that stabilized the HJ intermediate, or blocked recombina-tion completely.2,3 The activity of many of these peptides, such asWRWYCR (Supplementary data Fig. 1B), is not dependent uponinteractions with any of the proteins required for recombination.In fact, these peptides inhibit several mechanistically distinct tyro-sine recombinases,3,4 DNA cleavage and HJ resolution by vacciniavirus topoisomerase,5,6 and structurallyunrelatedHJ-processing en-zymes like RecG helicase and RuvABC resolvase.7 All of these activi-ties are based on the ability of the peptides to bind to protein-freeHJs, and with somewhat lesser affinity to other branched DNAsand replication forks.7 Because HJs are central intermediates in sev-eral DNA repair pathways,8 we reasoned that these peptides mayinterfere with DNA repair and thus have antibacterial activity. In-deed, minimal inhibitory concentration (MIC) values range from32 to 64 lg/ml in Gramnegative bacteria and 4 to 32 lg/ml in Grampositive bacteria, includingmethicillin resistant Staphylococcus aur-eus (MRSA).4 Evidence from genetic and biochemical studiesll rights reserved.</p><p>: +1 619 594 5676.gall).Microbiology, University of.</p><p>supports a model where the need for DNA repair causes the forma-tion of Holliday junctions that are trapped by the peptides; in thisway repair is blocked and the cells die.4,7,9,10</p><p>DNA repair is a novel antibacterial target. Moreover, target-siteresistance is unlikely to develop because HJs are generated inmulti-ple independent ways.8 Consistent with this hypothesis, we havebeen unable to isolate stable peptide resistant mutants. Due to theincreasing problem of multiple drug resistance in bacteria, wewanted to identify smallmoleculeswith similaractivities as thepep-tides.We performed screens of 11 smallmolecule libraries compris-ing over nine million individual compounds to identify functionalanalogs of the HJ trapping peptides. Here we present the identifica-tion and characterization of an N-methyl aminocyclic thiourea thatis an active inhibitor of our in vitro recombination assays, binds spe-cifically to protein-free HJs, and inhibits a HJ-processing helicase.</p><p>Compound identification. The mixture-based libraries wescreened were synthesized at Torrey Pines Institute for MolecularStudies (reviewed in Ref. 11). In general the compounds in thelibraries we tested had low molecular weights and three diversitypositions, orRgroups, built onunique scaffolds (Fig. 2 in Supplemen-tary data). These scaffolds were first evaluated for inhibition of site-specific recombination without regard to individual R groups in thelibraries by making a mixture of mixtures. For example, the 40 R1,37 R2, and 80 R3 mixtures of the N-methyl aminocyclic thiourea li-brary (DCR 528, Supplementary data Table 1) were pooled togethertomakeonemixture comprising the total diversityof that library (40R1 37 R2 80 R3 = 118,400 compounds). This mixture of</p><p></p></li><li><p>Figure 1. Scaffold ranking. (A) Lanes from TrisTricineSDS gels showing recombi-nation reactions in the presence of the small molecule mixtures at 10 lg/ml inbent-L recombination. Sub refers to the double radioactive end-labeled DNAsubstrate which recombines with an unlabeled partner to generate products, Prod.CPD refers to covalent proteinDNA complexes and HJ is the Holliday junction. (B)Quantitation of the reactions shown in Figure 2A for HJ accumulation (left Y-axis)and concurrent reduction in the formation of recombinant products (right Y-axis).Int and IHF are the proteins performing the recombination reaction.</p><p>4532 D. K. Ranjit et al. / Bioorg. Med. Chem. Lett. 20 (2010) 45314534mixtures was tested in Int-mediated recombination between twoattL sites known as the bent-L2,12 pathway at 10 lg/ml and quanti-tated with respect to accumulation of HJs (Fig. 1). Individual com-pounds from the mixture are present in the recombinationreactions at 84 pg/ml (225 pM for DCR 528 given an averagemolecular weight of 374 g/mol for compounds in that library). Thisconcentration is well below the IC50 of even our most potent peptideinhibitor. However, as discussed previously,2,11 the inhibitory activityseen in the libraries is likely the result of a number of chemically sim-ilar compoundswith characteristics and potency close to themost ac-tive inhibitors and this group increases the effective concentration ofthe inhibitors in themixtures. As seen in Figure 1, the accumulation ofHJs in treated reactions coincides with a reduction in recombinantproducts as catalysis in the context of the HJ is blocked (refer to Sup-plementary data, Fig. 1A). Of the libraries screened, the N-benzylaminocyclic thiourea scaffold (DCR 527) and the N-methyl aminocy-clic thiourea scaffold (DCR 528) accumulated the most HJs and inhib- The concentration of individual compounds (in lg/ml) that are present in thelibrary or in the defined mixtures are calculated from the final concentration of themixture in the treated reaction divided by the diversity of the mixture. For instance,treatments with library DCR 528 at 10 lg/ml gives a final compound concentration of84 pg/ml (10 lg/ml/118,400 compounds), and treatments with a defined DCR 528mixture (such as mixture 126) at 1 lg/ml gives a final compound concentration of337 pg/ml (1 lg/ml/2960 compounds).ited recombination to the greatest extent (Fig. 1B). Therefore, wepursued both libraries.</p><p>Following scaffold selection, defined R group mixtures werescreened to identify individual compounds with activity (reviewedin Refs. 11,13). Each mixture has a defined position and individualcompounds within that mixture all have the same functionality atthat R group position; the other two R group positions contain equi-molar mixtures of the other functionalities. For example, mixture126 in the N-methyl aminocyclic thiourea library (SupplementarydataTable1) contains thedefined functionalityN0N-dimethyl-S-pro-pylamine at position R1, and a mixture of the 37 R2 and 80 R3 func-tionalities at the other two positions. Thus, this single mixturecontains a total of 2960 (1 37 80) possible compounds, eachpresent at 337 pg/ml (900 pM) when tested in reactions at1 lg/ml, and this allows evaluation of the functionality N0N-di-methyl-S-propylamine at position R1 with respect to inhibition ofrecombination. Functionalities at R2 and R3 were screened in thesame fashion. The mixtures from DCR 528 (Fig. 3 in Supplementarydata) averaged twofold more HJ accumulation than DCR 527 (datanot shown) and thus we chose to identify compounds from DCR528. These top ranking mixtures are designated with an asterisk inSupplementary data, Figure 3, and each mixture was titrated moreextensively in the bent-L pathway and ranked based on potency.The top defined mixtures were also shown to inhibit attL attR(excisive12) recombination, albeit with lower potencies.2,14 In orderto test the specificity of thesemixtures, theywere assayed in severalother reactions involvingDNAtransactionsandwere foundnot to in-hibit restrictiondigests byNdeI andHindIII, or plasmid relaxationbyE. coli topoisomerase I at 10 lg/ml.14 The mixtures were also tested(as described in Ref. 4) at 25 lg/ml, and shown to inhibit the growthof S. aureus.14 Basedon thesedata, threeR1, threeR2, andoneR3 func-tionalitieswere selected and the combinations resulted in nine indi-vidual compounds (Table 1) thatwere synthesized as described.15,16</p><p>The crude compounds were characterized in several in vivo andin vitro assays similar to those used to assess the definedmixtures.14</p><p>Basedon thesedata TPI1530-1 (Fig. 2)was selected for further study.Compound characterization. Compound 1530-1 was purified to</p><p>&gt;95%andverifiedbyLC-MS.Wetitrated1530-1 into excision recom-binationreactions (Fig. 3A) anddetermined IC50 valuesof3 lg/ml foraccumulation of HJs, and 5 lg/ml for inhibition of recombination(compared with IC50 values for peptide WRWYCR of 0.04 and0.12 lg/ml, respectively; Table 2). We tested for whether 1530-1inhibits several restriction enzymes as well as whether it bindsnon-specifically to double stranded DNA (an attL site12 in the ab-sence of recombination enzymes). 1530-1 inhibited restriction di-gests at 100 lg/ml by 22% compared to untreated reactions (Table2); however, no evidence of binding to double stranded DNA alonewas detected at this concentration. This suggested specificity forthe common HJ intermediate in the recombination assays. To testthis further, 1530-1 was examined for its ability to bind to protein-free HJ substrates and shown to form a specific complex similar tothat seenwithpeptideWRWYCR (Fig. 3B).We then tested the effectsof the compound on RecG-mediated unwinding of HJs. RecG is amonomeric SFX family helicase that recognizes HJs and unwindsthese substrates in the presence of ATP andmagnesium.17 This heli-case bears no structural or mechanistic similarity to the proteins inthe recombination assays and has been used previously to examinethe specificity of our peptides for the HJ.7,10 As seen in Figure 3C,1530-1 does inhibit RecG activity in a dose dependent manner withan IC50 of 0.85 lg/ml (compared with 0.12 lg/ml for WRWYCR).These data further corroborate a model where the inhibitor recog-nizes the structure of the HJ intermediate.</p><p>The ability of 1530-1 to inhibit bacterial growth was examinedusing several Gram (+) and Gram () bacteria. In contrast to theactivity of the DCR 528 library, compound 1530-1 did not inhibitbacterial growth, even at the highest concentration tested</p></li><li><p>Table 1Deconvolution of positional scanning data for the N-methyl aminocyclic thiourea library</p><p>Compound R1 R2 R3</p><p>1530-1 N0N-Dimethyl-S-propylamine R-Hydroxy-methyl 2-(4-Nitro-phenyl)-ethyl1530-2 R-Hydroxy-methyl1530-3 S-3-Methyl-1-methyl-indole1530-4 N0N-Dimethyl-S-propylamine S-3-Methyl-1H-indole1530-5 R-Hydroxy-methyl1530-6 S-3-Methyl-1-methyl-indole1530-7 N0N-Dimethyl-S-propylamine S-Hydroxy-methyl1530-8 R-Hydroxy-methyl1530-9 S-3-Methyl-1-methyl-indole</p><p>Figure 2. 1530-1 is built on a N-methyl aminocyclic thiourea scaffold withfunctionalities N0N-dimethyl-S-propylamine, R-hydroxy-methyl, and 2-(4-nitro-phenyl)-ethyl (R1, R2, and R3, respectively).</p><p>D. K. Ranjit et al. / Bioorg. Med. Chem. Lett. 20 (2010) 45314534 4533(100 lg/ml, data not shown). Lack of inhibitory activity may be duepoor to permeability of 1530-1 into bacterial cells. To address thispossibility we tested a hyperpermeable Salmonella enterica serovarTyphimurium (Stm) strain with mutations in the galE and rfagenes; this strain has shorter lipopolysaccharide (LPS) chains inthe outer membrane and increased permeability to many com-pounds, including our peptides.4,1820 At 100 lg/ml, 1530-1 inhib-ited the growth of this mutant by 50% after 22 h of incubation (datanot shown). Alternatively, 1530-1 may be rapidly effluxed from thecell. To address this we tested an E. coli strain with a deletion of theFigure 3. 1530-1 interacts with Holliday junctions. All concentrations in lg/ml. (A) Doseproteins and Integration Host Factor (IHF). (B) 1530-1 causes a shift in the migration racontrol reactions with peptide WRWYCR. (C) 1530-1 inhibits RecG-mediated unwindingDNA to a replication fork. This activity is inhibited by 1530-1 in a dose dependent manacrAB genes, which inactivates the major drug efflux pump in thebacterium and increases susceptibility to many antibiotics.21</p><p>At 100 lg/ml of 1530-1 the growth of the efflux mutant was notinhibited (data not shown). These data suggest that 1530-1 mayhave antibiotic activity but that poor membrane permeability mayprevent the compound from entering cells and finding its target.14</p><p>In summary,we have identified andpurified anN-methyl amino-cyclic thiourea that recognizes HJs and inhibits enzymes that cata-lyze the resolution of these intermediates of DNA recombinationand repair. This compound is composed of a similar combinationof R-group functionalities as the side chains of the peptide inhibi-tors:3,6 aromatic and basic functionalities are present in both themost potent peptides and in the other N-methyl aminocyclic thio-ureas that were identified (Table 1). Molecular modeling corrobo-rated by fluorescence quenching studies on peptide WRWYCRsuggested that aromatic functionalities may be involved in bas...</p></li></ul>